Method and apparatus for radioimmunoassay with regeneration of immunoadsorbent

ABSTRACT

Method and apparatus for radioimmunoassay providing a continuously reusable short cycle analytical system of particular use in biochemical analysis such as the determination of steriods, polypeptides and the like. The method disclosed comprises admixing an unlabelled antigen sample with a known concentration of labelled antigen, bringing the mixture into contact with a mass of appropriate antibody that has been immobilized thereby to bind at least part of the mixed antigen to the antibody. The amount of bound and/or unbound labelled antigen is detected and counted and, by use of previously prepared standards, the unknown concentration of unlabelled antigen is found. The still immobilized antibody is regenerated for immediate reuse by rinsing it with a selected eluting solution that is capable of breaking the antigen-antibody bond without altering the characteristics of the antibody. Alternate ways of immobilizing the antibody are described. Several suitable eluting solutions are disclosed. Also disclosed is a novel apparatus by which the new method may be automatically carried out and the antibody reused many times. By proper substitution, the antibody and antigen may be reversed.

United States Patent [191 Johnson METHOD AND APPARATUS FORRADIOIMMUNOASSAY WITH REGENERATION OF IMMUNOADSORBENT Lavell R. Johnson,Salt Lake City, Utah [73] Assignee: Summa Corporation, Las Vegas,

Nev.

[22] Filed: Mar. 19, 1973 [2l] Appl. No.: 342,513

[75] Inventor:

Primary ExaminerBenjamin R. Padgett Attorney, Agent, or Firm-Mario A.Martella [4 1 July 22, 1975 [57] ABSTRACT Method and apparatus forradioimmunoassay provid ing a continuously reusable short cycleanalytical system of particular use in biochemical analysis such as thedetermination of steriods, polypeptides and the like. The methoddisclosed comprises admixing an unlabelled antigen sample with a knownconcentration of labelled antigen, bringing the mixture into contactwith a mass of appropriate antibody that has been immobilized thereby tobind at least part of the mixed antigen to the antibody. The amount ofbound and/or unbound labelled antigen is detected and counted and, byuse of previously prepared standards, the unknown concentration ofunlabelled antigen is found. The still immobilized antibody isregenerated for immediate reuse by rinsing it with a selected elutingsolution that is capable of breaking the antigen-antibody bond withoutaltering the characteristics of the antibody. Alternate ways ofimmobilizing the antibody are described. Several suitable elutingsolutions are disclosed. Also disclosed is a novel apparatus by whichthe new method may be automatically carried out and the antibody reusedmany times. By proper substitution, the antibody and antigen may bereversed.

10 Claims, 1 Drawing Figure 1 METHOD AND APPARATUS FOR RADIOIMMUNOASSAYWITH REGENERATION OF IMMUNOADSORBENT BACKGROUND OF THE INVENTION l.Field of the Invention This invention relates generally to theanalytical technique known as radioimmunoassay in which antigens arebound to specific antibodies and, through the use of tracers (labels)and predetermined behavior standards the concentration of antigen in asample is determined. More particularly, the invention relates toimproved methods and apparatus for radioimmunoassay in which ashort-cycle time, hence more rapid analysis is achieved, the antibodymass (immunoadsorbent) is regenerated to be reused indefinitely ratherthan being wastes and the entire operation is automated.

2. State of the Art Radioimmunoassay is an analytical technique whichdepends upon the competition (affinity) of antigen for antigen-bindingsites on antibody molecules. in practice, standard curves areconstructed from work on a plurality of samples each containing (a) thesame known concentration of labelled antigen, and (b) various, butknown, concentrations of unlabelled antigen. Antigens are labelled witha radioactive isotope tracer. The mixture is incubated in contact withan antibody, the free antigen is separated from the antibody and antigenbound thereto, and then, by use of a suitable detector, such as a gammaor beta radiation detector, the percent of either the bound or freelabelled antigens is determined. This procedure is repeated for a numberof samples containing various known concentrations of unlabelledantigens and the results plotted. The percent of bound tracer antigensis plotted as a function of the antigen concentration. Typically, as thetotal antigen concentration increases the relative amount of the tracerantigen bound to the antibody decreases. After the standard graph isprepared, it is thereafter used to determine the concentration ofantigen in samples undergoing analysis.

ln actual analysis, the sample in which the concentration of antigen isto be determined is mixed with a known amount of tracer antigen. Tracerantigen is the same antigen known to be in the sample but which has beenlabelled with a suitable radioactive isotope. The sample with tracer isthen incubated in contact with the antibody. Thereafter, it may becounted in a suitable detector which counts the free antigen remainingin the sample. The antigen bound to the antibody or immunoadsorbent mayalso be similarly counted. Then, from the standard curve, theconcentration of antigen in the original sample is determined.Afterwards, the antibody or immunoadsorbent mass is discarded.

In order to detect the percentage of antigen that is bound to theantibody (bound antigen) and/or the percentage that remains free orunbound it is necessary to first separate the sample into a fractioncontaining bound antigen and one containing only free antigen. Onecommon method for doing this is to add a dextran coated charcoal to themixture. The charcoal is allowed to adsorb the free antigen. Thecharcoal with adsorbed free antigen is then separated from the antibody(and bound antigen) by centrifugation. Another known procedure is to addto the mixture another antibody which selectively precipitates the firstantibody (with the bound antigen) thus leaving in solution only freeantigen. Classification into appropriate free and bound fractions isthen effected by separating the precipitate from the supernatant bycentrifugation or other suitable means. Some workers have resorted tothe technique of binding the antibody to the inner walls of a plasticvessel, filling the vessel with the antigen bearing sample, allowing itto stand for an incubatioin period that typically ranges from 4 to 72hours and then separating free antigen from bound antigen by drainingand rinsing the vessel leaving therein only the antibody and boundantigen. A more recently developed technique is to prepare theimmunoadsorbent by binding the antibodies onto an insoluble cross-linkeddextran. The immunoadsorbent and antigen bearing sample are incubatedthen the dextran with bound antigen is separated from the solution bysuitable means.

In all of the foregoing procedures, the percentage of labelled antigenin either or both the bound or free fractions is determined and thestandard curve used to determine the antigen concentration. Thereafter,the immunoadsorbent is discarded.

Although the foregoing radioimmunoassay techniques have proven to bevaluable tools and have gained widespread acceptance, they are still notall that are to be desired because the antibody (immunoadsorbent) isconsumed with each analysis hence must be discarded. Moreover, priorpractice is batch type and the several reagents are added to theantibody in test tubes in which the separate steps, such as incubation,rinsing and the like, are performed, thus resulting in a slow and costlyoperation.

SUMMARY OF THE INVENTION The present invention provides improved methodand apparatus for carrying out radioimmunoassay. In accordance with theinvention the immunoadsorbent (antibody) is repeatedly and rapidlyregenerated thus obviating the need and therefore the time and cost ofconstant replacement. According to the invention, steps are incorporatedby which the method may be carried on continuously in constantlyrepeating cycles thereby eliminating the expensive time-consuming batchoperation. A novel equipment arrangement for automating the method isalso provided.

The invention is predicated on the discoveries that (l) by forming theimmunoadsorbent mass as an immobilized mass of antibodies through whichthe antigen sample and other reagents may flow the procedure may besequentially carried out without resort to the use of several manualsteps and (2) the immobilized immunoadsorbent may be regenerated forrepeated reuse by the rinsing with a solvent or eluting solution havingparticular characteristics. That is to say, bound antigens are releasedso that the antibody may be washed clean of bound antigen and therebyregenerated for reuse without affecting the essential characteristic ofthe antibody mass such as its ability to permit repeated flow of antigensolution therethrough and its antigenbinding efficiency during a largenumber of cycles.

As used herein, the term immobilized antibody mass refers to a mass ofantibodies held in place in a liquid stream flow path in such a mannerthat the stream may flow over or through the mass while the latterremains in place.

A suitable antibody mass may be formed from solid surfaces such as glassor water insoluble polymers to which the antibodies are attached byco-valent bonds. A mass of such beads with attached antibodies issupported on a screen in a tube or other hollow column through which theantigen bearing sample and other reagents are flowed sequentially.Covalent coupling of antibodies or antigens to said carrier is known.(It is disclosed for instance in U.S. Pat. No. 3,652,76l

In accordance with this invention, antibodies, specific t the antigenunder analysis, are co-valently coupled to a solid support such asbeads, a mass of the beads is immobilized, an antigen bearing solutionis flowed therepast, the percentage of free labelled antigen remainingin the solution is detected, the bound antigen is releasedsimultaneously with regeneration of the antibody mass and the percentageof released antigen is also detected. The percentages are determinedwith reference to the total antigen in the incoming sam ple. As noted,detection is of the tracer or labelled antigen.

Release of bound antigen and concommitant regeneration or reactivationof the antibody mass is effected by rinsing it with a solvent or elutingsolution that breaks the bond between antigen and antibody, but does notbreak the co-valent bond between the antibody and its support. Moreover,the solvent must not alter either the flow characteristic or antigenaffinity of the antibody mass.

Another type of immobilized antibody mass can be formed by encapsulatingantibodies in a semipermeable membrane. A quantity of such microcapsulesis then held in place in a column through which the antigen-bearingsolution flows. The membranes are selected to block the outward passageof antibodies while permitting the free passage of antigens and theirsolvent in both directions.

In the case of membrane capsules, since there is no covalent bond, thereis no need to be concerned with its destruction by the regeneratingsolvent. However, the limitation must still be observed that the solventnot adversely affect the flow characteristic or adsorption efficiency ofthe antibody. It is possible to increase efficiency of the membranes bycoupling the antibodies to a soluble polymer thereby increasing themolecular weight of the combined antibody thus enabling the use of arelatively more permeable membrane.

In connection with flow characteristics in either system, the solventshould not cause swelling of the antibody support or the membrane wallto the point where flow of relatively small antigens is blocked or theloss of relatively larger antibodies through the wall occurs.

In summary then, the method of the invention comprises the steps ofproviding a mass of immobilized specific antibodies to selected antigen,holding such mass in a liquid flow path, preparing an antigen bearingsample by adding a known quantity of labelled antigen to a samplecontaining an unknown amount of the same antigen, flowing such samplealong said liquid flow path over and in contact with said immobilizedantibody, thereafter detecting the quantity of free labelled antigenremaining in the sample and/or that which is bound to the antibody.

The quantity of antigen bound to the antibody is determined by rinsingthe antibody with a particular solvent to release bound antigens thendetecting the antigens in the rinse solution.

The solvents found to meet the requirements of this invention have beenhydrophobic in character. Typical solvents are methyl, ethyl andisopropyl alcohols as well as dimethylformamide.

In its most essential form, the apparatus invention comprises a contactchamber having an inlet and outlet and adapted to accommodate the flowof a liquid stream therethrough, a mass of antibodies, means forimmobilizing said antibodies and holding them in position in saidcontact chamber in the path of antigen flow therethrough, a detectionchamber having an inlet and outlet, a detector associated with saidchamber for determining the quantity ofa given tracer flowing throughsaid chamber, conduit means for conductng liquid from the outlet of saidcontact chamber to the inlet of said detection chamber, a source ofsample solution containing both labelled and unlabelled antigen, asource of regenerating solvent, means for supplying separately andsequentially to the inlet of said contact chamber for flow therethrougha quantity of said sample and of said solvent, and means for controllingthe flow of said sample and solvent through said contact chamber andsaid detection chamber. Means are also provided for introducing aso-called scintillation cocktail into the system between the contactchamber and detector to flow through the latter and therein to convertradioactive decay impulses to light or photons for detection by a photomultiplier detector.

The required flow rates through the contact chamber and detector aredetermined emperically and are controlled by metering pumps. The flowsequence is controlled by valves. By proper timing the flow through thesystem may be continuous with the rates being selected to providesufficient time at each station to achieve the results, such as binding,releasing or counting, sought at each station.

In order that the invention may be more readily understood and carriedinto effect, reference is made to the accompanying drawing and thedescription thereof which are offered by way of illustration and not inlimitation of the invention, the scope of which is defined by theappended claims and equivalents thereof rather than by any illustrativedescription.

BRIEF DESCRIPTION OF THE DRAWINGS AND DESCRIPTION OF PREFERREDEMBODIMENT The FIGURE is a schematic diagram of apparatus embodying theinvention.

As illustrated, the system comprises a sample source 10, a source 11 ofbuffer rinse, a source 12 of regenerating solvent, a source 13 ofscintillation cocktail, a rinse water reservoir 14, a contact chamber 16filled with immobilized antibodies (immunoadsorbent) a mixing chamber17, and a detector chamber or coil 18 with detector 19 adjacent thereto.The foregoing components are connected together by a series of conduitsand flow directing valves which, together with metering pumps and atiming mechanism, regulate flow through the system.

Specifically, the sample source 10 connects via a conduit 21 to atwo-position valve 22 which in one position connects to a sample loop23. The sample loop terminates in a two-position valve 24 which in oneposition connects to a conduit 26 leading to another twoposition valve27 which in one position connects the conduit 26 and sample loop 23 toan aspirator pump 28 which discharges to waste. A rinse water inletconduit 29 also connects to the pump 28 via the alternate polition ofvalve 27 to keep the pump primed and rinsed when it is not drawingsample.

The displacement rinse flows from tank 11 to a twoposition valve 31which in one position connects through a pump 32 to a furthertwo-position valve 33 whence it connects either to a conduit 34 leadingto the valve 22 and sample loop 23 or to a conduit 36 leading to thecontact chamber. mixer and detector. A conduit 37 connects the valve 24to the main conduit 36. A branch conduit 38 connects to the main conduit36 at a junction between the contact chamber and the detector at alocation ahead of the mix chamber 17. A pump 39 is provided to move areagent from the tank 13 through the conduit 38.

A central timer, generally designated 41, is employed to controlsequencing of the valves and pumps. The timer may be custom assembledfor the job in accor dance with available technology hence, itsstructure need not be described in detail. It is sufficient to indicate,as is done in the drawing, that the valves, all of which areelectrically actuatable solenoid valves, and the metering pumps, all ofwhich are electric, are connected to the timer by suitable conductors42.

OPERATION OF THE SYSTEM At the start of a cycle rinse reagent from thetank 11 passes through valve 31, pump 32, valve 33 into and throughconduit 36. This rinses the system. Sample from tank is drawn by pump 28through the valves into and through the sample loop 23 until the loop isrinsed and filled whereupon the valves 22, 24, 27 and 33 switch to thealternate positions whereupon there is a continuous flow from tank 11through valve 31, pump 32, valve 33, valve 22, loop 23, valve 24 andconduit 37 to conduit 36. Liquid flowing from tank 11 thus displacessample from the loop into and through the contact chamber 16. When theflow has continued long enough to displace all sample from the loop, thevalves 22, 24 and 33 return to the initial positions shown in thedrawing. This isolates the sample loop.

After the sample has passed through the contact chamber 16 and thedetection coil, the valve 31 shifts to accept regenerating solvent fromthe tank 12 and directs it through pump 32 and valve 33 directly intoconduit 36 to flow through the contact chamber where it releases thebound antigens and carries them to the mixer and detector.Simultaneously, the immunoadsorbent in the contact chamber isregenerated.

The scintillation cocktail in tank 13 typically comprises a primaryscintillator, a secondary scintillator and a solubilizer all carried intoluene. its function is to mix with the antigen bearing solution in themix chamber 17 and then to convert the radioactive impulses of thetracer to light or photons which can be detected by a photo multiplierdetector. Photons may be detected by either a gamma or betascintillation spectrometer.

The tracer or label is selected from those conveniently available. Alabel of H Tritium may be used with a beta scintillator detector with acoincidence circuitry counter whereas an lodine label 1 may be used witha gamma detector which does not require the coincidence circuitry. Inthe tests reported herein, a tritium label was used.

It is important that the sample loop be kept uncontaminated with theregenerating solvent since traces of that mixed with the sampler couldreduce the bonding efficiency in the contact chamber.

Although a very elemental sample is illustrated, it will be appreciatedthat several sophisticataed samplers are readily available that may beadapted to use in this invention.

A series of basic tests were performed to confirm that immunoadsorbentswhen formed as immobilized antibodies could be consistently regeneratedwith suitable reagents.

Reagents used were as follows:

Buffer Rinse (Solution A) 0.02 M sodium phosphate, pH 7.5 0.05 M sodiumchloride 0.01% merthiolate 0.02% sodium azide Regenerating solvent(Solution 8) Solution A with the addition of 30% (v/v) of ethyl alcohol.Scintillating Solution (Solution C) Five parts of a solution of 0.4%(w/v) 2.5 diphenyloxazole 0.008% (w/v) l, 4-bis-2(4-methyl-5-phenyloxazolyl) benzene toluene as solvent one part of Scintisol-GP(manufactured by lsolab,

Inc.)

EXAMPLE I A contact chamber was prepared from a glass pipette plugged atone end with glass wool. A slurry of 0.04 ml of immunoadsorbentsuspension was introduced. The suspension contained 20 mg/ml of solidsupport to which testosterone antibody had been covalently bonded. Thechamber was washed with 5 ml of a buffer rinse (Solution A) then by 1 mlof regenerating solvent (Solution B) and finally by another 2 ml ofbuffer rinse (Solution A.) The cycle was repeated several times. Eachfraction discharged from the contact chamber was separately collected inscintillation vials mixed with 15 ml of scintillation cocktail (SolutionC) and counted for 5 minutes in a scintillation spectrometer. Theresults are shown in Table l in which for each of four cycles thepercent of bound antigen and percent of free antigen are tabulated. inthe case of the bound antigen, it was first released from theimmobilized antibody by the regenerating solvent then it was detectedand counted.

TABLE I (Sample "Pl-Testosterone) Cycle Cycle Cycle Cycle 1 2 3 4Percent free labelled Antigen 61.3 33.4 33.l 33.3 Percent Bound"Labelled Antigen 40.3 65.3 70.5 7 l .4

The first two cycles were used to stabilize conditions.

Measured after passage through the immobilized antibody.

"Measured after release from the immobilized antibody by theregenerating solvent (Solution B).

EXAMPLE II (62.nC/ml of tritium) plus a known amount of unlabelledestriol. The concentration in the first column recites only the knownunlabelled estriol concentration.

These two series were separated in time. "The bound fraction wasmeasured afier release by the regenerating solvent.

All runs were made on a single antibody mass. Twenty one actual clinicalsamples were run through the same mass, interspersed among the runsreported in Table ll. Additionally, several hundred more standards andother samples were run through the same antibody mass so that it hasactually been regenerated about 500 times without detrimental loss inantigen binding efficiency. The tests reported in connection with Tablell were all run on the operating prototype hereinafter described inExample [11.

A variety of regenerating solvents have been tested and foundsatisfactory for release of bound antigen and regeneration of antibody.Some solvents, particularly ethyl and isopropyl alcohols, havedemonstrated almost indefinite regeneration capacity. In fact, as notedwith Solution B, an antibody mass has been regenerated about 500 times.The required characteristics of the solvent are that it breaks theantibody-antigen bond without destroying the flow characteristics orantigen binding efficiency of the antibody. In practice it will benecessary empirically to determine the proper solvent for any givensystem.

EXAMPLE III A prototype system was constructed which employed aturntable sampler holding a plurality of samples and having a lifter formoving the sample tube 21 into and out of samples as they aresequentially rotated into position by a timer. A cyclic timer 41 wasemployed. A commercially available rotary sample valve was used toperform the functions of valves 22, 24 and 33 in response to signalsfrom the timer. The valves 27 and 31 were simple twopositionelectrically operated valves. Suitable metering pumps 28, 29 and 32 wereused. Flow lines were small bore Teflon plastic tubes typically of0.0012 to 0.0062 inch ID. The contact chamber consisted of apolypropylene tube 0. l 25 inch inside diameter by 0.188 inch long. Asused. the colums contained 0.04 ml of a Sephadex G-25 suspension at aconcentration of mg Sephadex/ml of a solution of 0.02M sodium phosphateat pH 7.5, 0.05 M NaCl, 0.01% merthiolate and 0.02% sodium oxide.Antibody to the antigen was covalently bonded to the Sephadex. Thesample loop 23 had a capacity of 0.2 ml. Flow rates were:

Through the sample loop 0.45 ml/minute for 2 minutes From tank 11 or 12via pump 32 0.1l2 ml/minute From tank 13 via pump 39 -l.0 ml/minute.

The coil forming the detector cell has a volume capacity of 2.2 ml. Themixer 17 has a capacity of approximately l ml. Detection and countingwas accomplished by means of a beta scintillation detector adjacent thecoil and coupled to a counter not shown but which is a convenientlyavailable type.

The immunoadsorbent in the contact chamber was an antibody coupled toSephadex beads which were in turn immobilized in the contact chamber bynylon screen (325-400 mesh). Sephadex is a cross-linked dextran made byPharmacia A.B. of Uppsala. Sweden. Such beads or other particulatesolids with the ability to hold antibodies by co-valent bonds makeconvenient supports on which to bind the antibodies for immobilization.

As previously mentioned the tests reported in connection with Table I]were all run in apparatus of the foregoing type.

In operation, a typical total cycle time for some tests was 28 minutes.This total included a three minute rinse cycle during which rinse fromtank ll flushed the system free from regeneration solvent. The sampleloop, which does not require preliminary rinse, is at the same timeflushed and filled with sample. The first three minute period isfollowed by a ten minute period during which reagent from tank 11 flowsthrough the sample loop displacing sample therefrom into and through thecontact chamber, the mixer and the detector. All of this is followed bya [5 minute regeneration period during which solvent from tank 12,by-passing the sample loop as previously described, flows through thecontact chamber to release bound antigens thereby regenerating theimmunoadsorbent. The released antigens are subjected to detection asthey move through the flow cell to eventual discard.

In other tests, the cycle time has been reduced to less than fifteenminutes. In general, the cycle time can be reduced by increasing theconcentration of radioactive tracer.

As noted, a suitable reagent for regeneration of the immunoadsorbent isone which will break the bond between the antigen and antibody but doesnot adversely affect the antibody. That is, it does not loosen it fromits support nor reduce either its permeability or affinity for antigen.So far identification of suitable reagents has been empirical on thebasis of behavior. However, once a suitable reagent for a givenantibody-antigen system has been identified, it becomes a permanentreagent for that system.

Thus, the unexpected discovery that regeneration is possible and theidentification of suitable reagents makes possible rapid analysis atgreatly reduced costs. Moreover, the system is capable of automationthereby reducing human error with a concomitant increase in accuracy.

Throughout the specification reference has been made to bound antigen.This refers to the antigen bound to the antibody mass. The bound antigenis mea sured only after it has been released from the antibody by theregenerating solvent. if the percent recovery of bound (released)antigen is added to the percent of free antigen measured in any cycle,the total is consistently at for practical purposes. This is significantbecause it confirms that substantially all bound antigen is releasedfrom the antibody and that the antibody is completely regenerated.

Although the invention has been described with particular reference tosteriods. it is not intended to be so limited as it has been used withsystems for the determination of polypeptides, thyroid hormones and someviruses. It is clear then, that the invention has broad application toradioimmunoassay procedures.

I claim:

1. In an analytical method for biochemical analysis of an antigenwherein a mass of antibodies specific to a selected antigen isimmobilized to form an immunoadsorbent and wherein a solution containingthe selected antigen is flowed into and out of contact with theimmunoadsorbent whereby at least part of said selected antigen is boundto said mass of antibodies immobilized by said immunoadsorbent and theremainder is not bound to said antibodies. the improvement whichcomprises the steps of:

regenerating the immunoadsorbent for reuse by releasing the antigenbound to the antibodies of the immunoadsorbent, said regenerating stepincluding contacting said immunoadsorbent with a solvent characterizedby the ability to break the bond between the antigen bound to theantibody of the immunoadsorbent to free substantially all of saidantigen therefrom without substantially adversely affecting theflowthrough quality of said immunoadsorbent and the capacity thereof tobind antigen material subsequently brought into contact therewith,utilizing at least one of l) the said remainder which is not bound tosaid aantibodies or (2) the antigen formerly bound to saidimmunoadsorbent and released during regeneration thereof, or both, in ananalytical assay of said selected antigen, and

repetitively reusing said regenerated immunoadsorbent for analyticalassays of further selected antigen material.

2. In an analytical method as set forth in claim I wherein theimmunoadsorbent is formed by co-valently bonding the said mass ofantibodies to individual solids of a mass of particulate solids,

said mass of solids being supported on a porous memher, and

said solvent is flowed through said mass.

3. In an analytical method as set forth in claim 1 wherein said mass ofantibodies is co-valently bonded to a cross-linked dextran polymer.

4. In an analytical method as set forth in claim I wherein theantibodies are specific to an antigen selected from the class consistingof steroids, polypeptides and thyroid hormones.

5. In an analytical method as set forth in claim 2 wherein theantibodies are specific to an antigen selected from the class consistingof steroids and thyroid hormones, and

said solvent comprising an alcohol.

6. In a radioimmunoassay analytical procedure in which a sample solutioncontaining antigen to be assayed is mixed with a known concentration oflabelled antigen, the mixed sample solution being brought into contactwith an immunoadsorbent formed by immobilizing a mass of antibodiesspecific to said antigen thereby to effect binding of part of thelabelled and unlabelled antigens in said mixed sample solution to theantibodies immobililzed by said immunoadsorbent. the remaining portionof the labelled and unlabelled antigens in said mixed sample solutionpassing through said immunoadsorbent and forming an unbound antigenfraction, the improvement which comprises:

regenerating the immunoadsorbent by releasing substantially all of theantigens bound to the im 5 munoadsorbent by rinsing said immunoadsorbentwith a solvent to break the bond between the antigens bound to saidantibodies which are immobilized by said immunoadsorbent thus forming(l) a released solvent fraction containing formerly bound labelled andunlabelled antigens and. (2) a regenerated immunoadsorbent substantiallyfree of bound antigens. detecting the labelled antigens in at least oneor the other or both of (a) said unbound antigen fraction and (b) saidreleased solvent fraction, as a function is of the quantity the antigento be assayed. and

thereafter reusing the said regenerated immunoad sorbent for analyticalassays of further sample solutions containing antigen as to which saidimmobilized antibodies are specific.

7. In a radioimmunoassay procedure as set forth in claim 6 wherein saiddetecting step includes detecting the percentage of labelled antigens insaid unbound fraction,

detecting the percentage of labelled antigens in said released solventfraction, and

determining the percentage of unlabelled antigens with reference to thetotal detected labelled antigens.

8. In a radioimmunoassay procedure as set forth in claim 6 wherein saidantibodies are specific to an antigen selected from the class consistingof steroids, polypeptides, thyroid hormones, and viruses, and

said solvent being hydrophobic in character.

9. In a radioimmunoassay procedure as set forth in claim 6 in which saidmixed sample solution is flowed into contact with and through saidimmunoadsorbent to effect binding a part of the labelled and unlabelledantigens,

detecting the labelled antigens in said unbound antigen fraction,

regenerating said immunoadsorbent and forming said released solventfraction,

detecting the labelled antigen in said released solvent fraction, and

reusing said regenerated immunoadsorbent by repeating the steps offlowing another mixed sample solution into contact therewith followed bydetecting the labelled antigen in the unbound fraction, regenerating theimmunoadsorbent, and detecting the labelled antigen in the releasedsolvent fraction.

10. In a radioimmunoassay procedure as set forth in claim 6 wherein saidimmunoadsorbent is formed by co-valently bonding said mass of antibodiesto polymeric material, and wherein said procedure is continuous andinvolves repetitively for different samples the recited steps of a.bringing a mixed sample solution into contact with the immunoadsorbentto form an unbound antigen fraction while binding a part of the labelledand unlabelled antigens on the immobilized antibodies,

b. regenerating the immunoadsorbent to form a released solvent fractionand to form a regenerated immunoadsorbent. and

c. detecting the labelled antigens in at least one or the other or bothof the unbound antigen fraction and said released solvent fraction.

II F l i i

1. IN AN ANALYTICAL METHOD FOR BIOCHEMICAL ANALYSIS OF AN NTIGEN WHEREINA MASS OF ANTIBODIES SPECIFIC TO A SELECTED ANTIGEN IS IMMOBILIZED TOFORM AN IMMUNOADSORBENT AND WHEREIN A SOLUTION CONTAINING THE SELECTEDANTIGEN IS FLOWED INTO AND OUT OF CONTACT WITH THE IMMUNODSORBENTWHEREBY AT LEAST PART OF SAID SELECTED ANTIGEN IS BOUND TO SAID MASS OFANTIBODIES IMMOBILIZED BY SAID IMMUNOADSORBENT AND THE REAMINDER IS NOTBOUND TO SAID ANTIBODIES, THE IMPROVEMENT WHICH COMPRISES THE STEPS OF:REGENERATING THE IMMUNOADSORBENT FOR REUSE BY RELEASING THE ANTIGENBOUND TO THE ANTIBODIES OF THE IMMUNOADSORBENT, SAID REGENERATING STEPINCLUDING CONTACTING SAID IMMUNOADSORBENT WITH A SOLVENT CHARACTERIZEDBY THE ABILITY TO BREAK THE BOND BETWEEN THE ANTIGEN BOUND TO THEANTIBODY OF THE IMMUNOADSORBENT TO FREE SUBSTANTIALLY ALL OF SAIDANTIGEN THEREFROM WITHOUT SUBSTANTIALLY ADVERSELY AFFECTING THEFLOW-THROUGH QUALITY OF SAID IMMUNOADSORBENT AND THE CAPACITY THEREOF TOBIND ANTIGEN MATERIAL SUBSEQUENTLY BROUGHT INTO CONTACT THEREWITH,UTILIZING AT LEAST ONE OF (1) THE SAID REMAINDER WHICH IS NOT BOND TOSAID ANTIBODIES OR (2) THE ANTIGEN FORMERLY BOUND TO SAIDIMMUNOADSORBENT AND RELEASED DURING REGENERATION THEREOF, OR BOTH, IN ANANALYTICAL ASSAY OF SAID SELECTED ANTIGEN, AND REPETITIVELY REUSING SAIDREGENERTED IMMUNOADSORBENT FOR ANALYTICAL ASSAYS OF FURTHER SELECTEDANTIGEN MATERIAL.
 2. In an analytical method as set forth in claim 1wherein the immunoadsorbent is formed by co-valently bonding the saidmass of antibodies to individual solids of a mass of particulate solids,said mass of solids being supported on a porous member, and said solventis flowed through said mass.
 3. In an analytical method as set forth inclaim 1 wherein said mass of antibodies is co-valently bonded to across-linked dextran polymer.
 4. In an analytical method as set forth inclaim 1 wherein the antibodies are specific to an antigen selected fromthe class consisting of steroids, polypeptides and thyroid hormones. 5.In an analytical method as set forth in claim 2 wherein the antibodiesare specific to an antigen selected from the class consisting ofsteroids and thyroid hormones, and said solvent comprising an alcohol.6. In a radioimmunoassay analytical procedure in which a sample solutioncontaining antigen to be assayed is mixed with a known concentration oflabelled antigen, the mixed sample solution being brought into contactwith an immunoadsorbent formed by immobilizing a mass of antibodiesspecific to said antigen thereby to effect binding of part of thelabelled and unlabelled antigens in said mixed sample solution to theantibodies immobililzed by said immunoadsorbent, the remaining portionof the labelled and unlabelled antigens in said mixed sample solutionpassing through said immunoadsorbent and forming an unbound antigenfraction, the improvement which comprises: regenerating theimmunoadsorbent by releasing substantially all of the antigens bound tothe immunoadsorbent by rinsing said immunoadsorbent with a solvent tobreak the bond between the antigens bound to said antibodies which areimmobilized by said immunoadsorbent thus forming (1) a released solventfraction containing formerly bound labelled and unlabelled antigens and,(2) a regenerated immunoadsorbent substantially free of bound antigens,detecting the labelled antigens in at least one or the other or both of(a) said unbound antigen fraction and (b) said released solventfraction, as a function of the quantity the antigen to be assayed, andthereafter reusing the said regenerated immunoadsorbent for analyticalassays of further sample solutions containing antigen as to which saidimmobilized antibodies are specific.
 7. In a radioimmunoassay procedureas set forth in claim 6 wherein said detecting step includes detectingthe percentage of labelled antigens in said unbound fraction, detectingthe percentage of labelled antigens in said released solvent fraction,and determining the percentage of unlabelled antigens with reference tothe total detected labelled antigens.
 8. In a radioimmunoassay procedureas set forth in claim 6 wherein said antibodies are specific to anantigen selected from the class consisting of steroids, polypeptides,thyroid hormones, and viruses, and said solvent being hydrophobic incharacter.
 9. In a radioimmunoassay procedure as set forth in claim 6 inwhich said mixed sample solution is flowed into contact with and throughsaid immunoadsorbent to effect binding a part of the labelled andunlabelled antigens, detecting the labelled antigens in said unboundantigen fraction, regenerating said immunoadsorbent and forming saidreleased solvent fraction, detecting the labelled antigen in saidreleased solvent fraction, and reusing said regenerated immunoadsorbentby repeating the steps of flowing another mixed sample solution intocontact therewith followed by detecting the labelled antigen in theunbound fraction, regenerating the immunoadsorbent, and detecting thelabelled antigen in the released solvent fraction.
 10. In aradioimmunoassay procedure as set forth in claim 6 wherein saidimmunoadsorbent is formed by co-valently bonding said mass of antibodiesto polymeric material, and wherein said procedure is continuous andinvolves repetitively for different samples the recited steps of a.bringing a mixed sample solution into contact with the immunoadsorbentto form an unbound antigen fraction while binding a part of the labelledand unlabelled antigens on the immobilized antibodies, b. regeneratingthe immunoadsorbent to form a released solvent fraction and to form aregenerated immunoadsorbent, and c. detecting the labelled antigens inat least one or the other or both of the unbound antigen fraction andsaid released solvent fraction.